Thirty temperature-sensitive mutants of encephalomyocarditis (EMC) virus were previously isolated and partially characterized. Several of these mutants have a similar defects in the proteolytic processing of the capsid protein precursors when grown at the nonpermissive temperature of 39.5 degrees C. With a cell-free translation system using a rabbit reticulocyte lysate, we found that the mutants are not defective in the viral-specific protein involved in proteolytic cleavage, but, rather, defective in the capsid precursor proteins to be cleaved. We propose also to study mutants of EMC virus which are phenotypically defective in the synthesis of RNA at the nonpermissive temperature (RNA minus mutants). Since it has been proposed that the viral protease may regulate RNA polymerase activity, some RNA minus mutants may potentially be defective not in the RNA polymerase but in the viral protease. Several cleavage-defective mutants have defects in the capsid precursor proteins which prevent the proper assembly of virus at the nonpermissive temperature. We propose to examine the subassembly structures which likely accumulate at the nonpermissive temperature in cells infected with cleavage-defective mutants and learn more of the viral assembly process. We have characterized many of the EMC wild type virus polypeptides seen in purified virus and in infected cells with two-dimensional electrophoresis. All of the proteins from purified virus can be resolved by two-dimensional gel electrophoretic techniques. All of the known stable virus polypeptides seen in wild-type infected cells have been resolved by two-dimensional gel electrophoresis but the primary and intermediate precursor proteins cannot be resolved. We are attempting to resolve these proteins with alternative procedures. In addition, we are continuing to examine the viral polypeptides in mutant virus infected cells with two-dimensional gel electrophoresis to determine if mutant viral proteins contain differences in charged amino acids relative to wild type viral proteins.